Molecular cloning has become a powerful tool for the amplification of specific DNA (deoxyribonucleic acid) fragments and their subsequent isolation in high yields. Two basic steps are involved in molecular cloning. First the DNA fragments to be cloned are joined in vitro to an autonomously replicating cloning vehicle molecule, e.g., plasmid DNA [Cohen, S. N. et al., PNAS 70, 3240 (1973); Tanaka, T. and Weisblum, B., J. Bacteriology 121, 354 (1975)] or .lambda. phage DNA [Thomas, M. et al., PNAS 71, 4579 (1974); Murray, N. E. and Murray, K., Nature 251, 476 (1974)]. The hybrid recombinant DNA-cloning vehicles so formed are then introduced into host cells, e.g., E. coli cells, by transformation and cloned by a suitable technique such as single colony isolation or plaque formation.
In one cloning method, two different DNA molecules are cut by the same restriction endonuclease to produce identical cohesive ends. The DNA molecules are annealed to one another and then covalently joined by DNA ligase. This method limits the size and kind of DNA fragments that can be cloned since it often requires cloning of a much larger DNA fragment that one is inserted in. For example, if one wants to clone a small DNA fragment such as a promoter (e.g., an RNA polymerase protected fragment), the nearest restriction endonuclease sites may be relatively distant, and thus extraneous DNA sequences must be included in the cloned DNA. This creates the possibility that undesirable or even hazardous sequences may be transferred, and it is this possibility which has led to public interest in the entire area of molecular cloning and recombinant DNA research. Furthermore, many DNA fragments cannot be cloned by this method because of the lack of a suitable restriction enzyme for producing molecules with appropriate cohesive ends.
The present invention utilizes chemically synthesized oligonucleotides having nucleotide sequences which are the recognition sites for restriction endonucleases as adaptor molecules. These adaptor molecules are joined at the ends of natural or synthetic DNA molecules to form adapted DNA molecules. The ends of such natural or synthetic DNA molecules can be even-ended or can have a protruding nucleotide sequence. Alternatively, adapted DNA molecules which comprise synthetic DNA molecules having such adaptor molecules incorporated therein at their ends are prepared. Such adapted DNA molecules are then joined to a cloning vehicle, thereby making the cloning procedure much more selective, versatile and safe. A part of the substance of this invention has been described recently in two publications (Bahl, Chander P., et al., Gene 1, 81 (1976) and Marians, K. J. et al., Nature, 263, 744 (1976)]. These publications are hereby incorporated into the present disclosure and made part thereof.